Cell-cell communication between fiber cells is one of the most striking aspects of lens cell organization and is critical for the normal physiology of this tissue, which is devoid of vasculature. Alterations in such communication result in cataract formation both in human and mouse. In our preliminary experiments we characterized a new pathway, which is permeable to macromolecules and form in the core of the lens during development. This pathway is distinct from gap junctions and interconnects fiber cells in the lens core to form a true syncytium. The purpose of this Proposal is to characterize the developmental properties of the core syncytium, elucidate physiological significance and molecular mechanism of the syncytium formation in various vertebrate species, including humans. We will address four questions: 1) Is core syncytia a feature of all vertebrate lenses? We will approach the answer by characterization of syncytia in chicken, mouse and primate lenses, testing spatio-temporal correlation of syncytium formation with organelle loss and denucleation both in embryonic and adult lenses. We will perform morphometric and functional analysis to determine developmental onset time of the syncytium in the mouse. 2) What is the function of the lens core syncytium? To define the function we will test whether native lens proteins are able to translocate from biosynthetically active peripheral fiber cells into quiescent cells of the core region within the syncytium. We will monitor translocation of pulse-labeled proteins and determine which protein species use this communication pathway. 3) Does fusion pore formation represent a mechanism for protein-permeable cell coupling within lens core syncytium? We will test whether fusion formation between fibers always precedes syncytial formation in mouse and chicken embryos. We will use ultrastructural analysis to determine density and normal size variation of fusion pores, connecting fiber cells within syncytium to map pore distribution across the lens. 4) What is the molecular mechanism, underlying fiber cell fusion? We will test abundant membrane proteins ADAM12, Cx46, Cx50 and AQP0 for the role in cell fusion and syncytium formation in the lens. We will examine connexin knockout mice and transgenic mice, expressing dominant negative or fusogenic form of ADAM12, for the presence of syncytium and cell fusions in the lens core. We will also test whether ADAM12, Cx46, Cx50 and AQP0 co-localize to each other and to the fusion pores, coupling lens fiber cells. This research project will help reveal new aspects of lens development, aging and cataract formation.